WO2007149433A2 - Tumor-specific delivery of therapeutic agents via liposomase - Google Patents

Tumor-specific delivery of therapeutic agents via liposomase Download PDF

Info

Publication number
WO2007149433A2
WO2007149433A2 PCT/US2007/014274 US2007014274W WO2007149433A2 WO 2007149433 A2 WO2007149433 A2 WO 2007149433A2 US 2007014274 W US2007014274 W US 2007014274W WO 2007149433 A2 WO2007149433 A2 WO 2007149433A2
Authority
WO
WIPO (PCT)
Prior art keywords
biological agent
tumor drug
composition
tumor
seq
Prior art date
Application number
PCT/US2007/014274
Other languages
English (en)
French (fr)
Other versions
WO2007149433A3 (en
Inventor
Ian Cheong
Shibin Zhou
Kenneth W. Kinzler
Bert Vogelstein
Original Assignee
The Johns Hopkins University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Johns Hopkins University filed Critical The Johns Hopkins University
Priority to KR1020087031596A priority Critical patent/KR101376634B1/ko
Priority to CN200780027001.3A priority patent/CN101489593B/zh
Priority to JP2009516542A priority patent/JP5319523B2/ja
Priority to US12/304,178 priority patent/US8444963B2/en
Publication of WO2007149433A2 publication Critical patent/WO2007149433A2/en
Publication of WO2007149433A3 publication Critical patent/WO2007149433A3/en
Priority to US13/692,064 priority patent/US8568708B2/en
Priority to US13/963,387 priority patent/US8901100B2/en
Priority to US14/556,890 priority patent/US20150306184A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1277—Preparation processes; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01003—Triacylglycerol lipase (3.1.1.3)

Definitions

  • This invention is related to the area of oncology. In particular, it relates to treatment of tumors which enhance the selective toxicity of anti-cancer agents.
  • the third strategy (angiogenic) exploits abnormal aspects of tumor vasculature with agents such as Avastin (5, 6) or drugs incorporated into liposomes (7).
  • Liposomes are relatively large particles that can penetrate through the fenestrated endothelium present in tumors and a few other organs (8, P). Once they gain access to tumors, they persist and eventually release their contents and raise local drug concentrations through the enhanced permeabilization and retention (EPR) effect (JO).
  • EPR enhanced permeabilization and retention
  • JO enhanced permeabilization and retention
  • a composition useful for delivering therapeutic agents.
  • the composition comprises toxin-defective Clostridium novyi spores; and a liposome comprising an anti-tumor drug or biological agent.
  • composition comprises a toxin-defective Clostridium novyi liposomase according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif (residues 160-164 of SEQ ID NO: 1) and a liposome comprising an anti-tumor drug or biological agent.
  • kits comprises a toxin-defective
  • kits comprises toxin-defective Clostridium novyi spores; and a liposome comprising an anti-tumor drug or biological agent.
  • Yet another embodiment of the invention is a method of treating a cancer patient.
  • a first agent which is toxin-defective Clostridium novyi spores and a second agent • which is a liposome comprising an anti-tumor drug or biological agent are administered to the cancer patient.
  • the tumor thereby regresses or its growth is slowed or arrested.
  • An additional aspect of the invention is a method of treating a cancer patient in which a first agent and a second agent are administered to the cancer patient.
  • the first agent is a toxin-defective Clostridium novyi liposomase according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif
  • the second agent is a liposome comprising an anti-tumor drug or biological agent.
  • the tumor thereby regresses or its growth is slowed or arrested.
  • the present invention is a method of treating a cancer patient in which a first and a second agent are administered to the cancer patient.
  • the first agent is a vector encoding toxin-defective Clostridium novyi liposomase according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif
  • a second agent is a liposome comprising an anti-tumor drug or biological agent. The tumor thereby regresses or its growth is slowed or arrested.
  • the present invention also provides a composition comprising an isolated and purified toxin-defective Clostridium novyi liposomase protein according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif.
  • the invention further provides a conjugate protein comprising a toxin-defective Clostridium novyi liposomase protein according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif; and a polypeptide Iigand which binds to a receptor on a tumor cell.
  • a polynucleotide which encodes the conjugate protein is also provided.
  • the invention still further provides a composition comprising an isolated and purified polynucleotide encoding a toxin-defective Clostridium novyi liposomase protein according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif.
  • a composition comprising an isolated and purified polynucleotide encoding a toxin-defective Clostridium novyi liposomase protein according to SEQ ID NO: 1 or according to SEQ ID NO: 1 with a substitution mutation in the GXSXG lipase motif.
  • Fig. 1A-1D Effects of treatment with C. novyi-NT plus Doxil. Mice bearing the indicated tumors were treated on day 0 with various combinations of the indicated agents. Free doxorubicin plus C. novyi-NT spores resulted in deaths of all animals within two weeks and is not shown. Means and standard errors of data collected from at least five mice per group are illustrated. The differences between C. novyi-NT plus Doxil and the other groups were significant (p ⁇ 0.0006, log-rank test).
  • Figs. IA and IB show effect on CT26 tumors.
  • Figs. 1C and ID show effect on HTl 16 tumors.
  • Figs. IA and 1C show tumor volume and Figs. IB and ID show percent survival.
  • Fig. 2A-2B Pharmacokinetic distribution of Doxil after treatment with C. novyi-NT.
  • Fig. 2A At 0 hours, Doxil was administered to athymic nude mice bearing tumors of ⁇ 300 mm 3 in size. Another group of mice was intravenously injected with C. novyi- NT spores 16 hours prior to Doxil. Mice were sacrificed at the indicated time points and doxorubicin extracted from tissues and measured by fluorometry. Means and standard deviations from three mice per time point are shown.
  • FIG. 2B Tumors were snap-frozen 16 hr after injection with Doxil and cryostat sections from the central regions examined using a fluorescence microscope.
  • the fluorescence signal from doxorubicin in liposomes is quenched (see Supplementary Materials and Methods), while released doxorubicin emits fluorescence at 590 nm. Colocalization of the doxorubicin fluorescence and nuclear DNA, stained with DAPI, is observed in the tumors treated with C. novyi-NT spores.
  • FIG. 3A-3C Biochemical purification and Identification of Liposome-Disrupting activity.
  • C. novyi-NT was grown in medium until late log phase and the medium cleared of cells through centrifugation. Following precipitation with 40% saturated ammonium sulfate, ion exchange chromatography was performed and fractions evaluated for liposome-disrupting activity.
  • Fig. 3B The peak fractions (30 — 31) from (Fig. 3A) were pooled and fractionated by gel filtration chromatography.
  • FIG. 3C The peak fractions (29 - 30) from (Fig. 3B) were fractionated by SDS- polyacrylamide gel electrophoresis.
  • Fig. 4A-4D Functional analysis of liposomase. Plasmids carrying the wt or mutant forms of the NTOl CX2047 gene were introduced into E. coli. "Cured" bacteria represent those originally containing the wt gene and grown in the absence of selective antibiotics until the plasmid was lost. Cellular lysates from three independent clones of each bacterial strain were generated after induction of expression by IPTG and used for the following: (Fig. 4A) Western blotting with an antibody to oligohistidine confirmed the presence of similar amounts of wt and mutant proteins in the bacteria. (Fig. 4A)
  • FIG. 5 Effects of treatment with C. novyi-NT plus liposomal CPT-I l. Mice bearing the indicated tumors were treated on day 0 with various combinations of the indicated agents. Means and standard errors of data calculated from at least five mice per group are shown. The differences between C. novyi-NT plus Doxil and the other groups were significant (p ⁇ 0.0003, log-rank test).
  • Fig. 6A-6B Liposome-disrupting activity.
  • Fig. 6A Time course of doxorubicin release from Doxil in the presence of growth medium from C novyi-NT cultures ("conditioned medium"). C. novyi-NT was grown until late log phase and the medium cleared of cells via centrifugation. Significant liposome-disrupting activity was noted (red symbols) while no increase in fluorescence was observed in the indicated controls.
  • Fig. 6B Liposome-disrupting activity as a function of growth time in culture. C. novyi-NT spores were inoculated in growth medium and harvested at various times thereafter.
  • liposome-disrupting activity was measured in the supernatant and correlated with absorbance at 600 nm. Means and standard deviations of data from at least two independent assays of liposome-disrupting activity are shown. Standard deviations are not visible when the bars representing them are smaller than the symbol of the corresponding data point.
  • the inventors have developed methods for treating tumors.
  • the methods enhance the tumor specificity of treatments, thereby reducing systemic toxicities.
  • Using a combination of a protein made by C. novyi-NT and liposome encapsulated drugs or biological agents the efficacy of tumor elimination is greatly increased.
  • the protein while having lipase enzymatic activity appears to function non-ehzymatically as a liposome disrupter.
  • the lipase enzyme activity is not necessary for the liposome disrupting activity.
  • both wild-type and lipase-negative mutants can be used for this function.
  • the lipsome-disrupting function can be delivered by the protein as elaborated in situ by live C. novyi-NT, or by a cell-free preparation of the protein itself.
  • the protein can be delivered as part of a conjugate or fusion protein which provides additional desirable functionality to the liposomase protein.
  • chemotherapeutic drug or biological agent that can be encapsulated in a liposome.
  • drugs of the following categories include drugs of the following categories as examples: topoisomerase inhibitors, DNA synthesis inhibitors, cell division inhibitors, angiogenesis inhibitors, and microtubule inhibitors.
  • Antibodies and antibody conjugates such as rituxan, herceptin and erbitux, can also be encapsulated in a liposome.
  • cytokines and other bioactive proteins which may enhance the patient's endogenous tumor-fighting systems may be used.
  • cytokines can include, without limitation: IL-2 and interferon-alfa 2b and GM-CSF.
  • Particular drugs, cytokines, and antibodies which can be used without limitation include: abarelix; aldesleukin; Alemtuzumab; alitretinoin; allopurinol; altretamine; amifostine; anakinra; anastrozole; arsenic trioxide; asparaginase; azacitidi ⁇ e; BCG Live; bevacizumab; bexarotene capsules; bexarotene gel; bleomycin; bortezombi; bortezomib; busulfan; calusterone; capecitabine; carboplatin; carmustine; celecoxib; cetuximab; chlorambucil; cisplatin; cladribine; clofarabine; cyclophosphamide; cytarabine; dacarbazine; dactinomycin, actinomycin D; dalteparin sodium; darbepoetin
  • the gene encoding the liposomase has a sequence as shown in accession no. CP000382 of GenBank (SEQ ID NO: 2).
  • the protein has a sequence as shown in accession no. ABK60711 (SEQ ID NO: 1).
  • the first 35 amino acids are predicted to be cleaved as a signal sequence.
  • Mutants having amino acid substitutions in the highly-conserved GXSXG lipase motif also retain liposomase activity and can be used.
  • the S127G mutant (mutation at serine 162 in SEQ ID NO: 1) can be used to provide liposomase activity.
  • Serine-127 can be with amino acids A, C, D, E, F, G, H 5 1, K, L, M, N, P, Q, R, T, V, W, or Y.
  • compositions according to the invention can be made before or after administration to a human or other mammal.
  • components of the compositions can be administered separately or mixed. If separately, the components can be administered in any order.
  • the components may form the composition when they are within a patient or other mammal.
  • the components of the compositions can be packaged together or separately in a kit. Thus multiple vessels or containers within a single package may be provided to an end-user. The end user may administer the components separately or mixed, at one time or at more then one time.
  • Kits comprising the useful components for practicing the anti-tumor methods of the present invention can be packaged in a divided or undivided container, such as a carton, bottle, ampule, tube, etc.
  • the spores, liposomase, and anti-tumor agents can be packaged in, e.g., dried, lyophilized, or liquid form. Additional components provided can include vehicles for reconsititution of dried components. Preferably all such vehicles are sterile and apyrogenic so that they are suitable for injection into a mammal without causing adverse reactions.
  • the anti-tumor agents other than the spores are also preferably sterile.
  • the spores are preferably microbiologically pure, i.e., containing no other bacteria other than the desired spore-forming anaerobe.
  • C. novyi spores can be prepared as is known in the art.
  • the spores will be from a toxin-defective strain. See for example, U.S. Patent Application 20050079157, the contents of which are expressly incorporated herein.
  • Toxin can be eliminated by a process of curing of a bacteriophage or plasmid.
  • a C. novyi useful as a starting material (ATCC 19402) can be obtained from the American Type Culture Collection, 10801 University Boulevard, Manassas, VA, 20110-2209.
  • strains need not be totally non-toxigenic, it is desirable that at least one of the toxin genes be mutated, deleted, or otherwise inactivated to render the bacteria less harmful to the host. If a toxin gene is episomal or on a phage, then curing of the episome or phage can be used to eliminate the toxin gene. Techniques are well known in the art for mutagenesis and screening of mutants and for curing episomes.
  • Isolated and bacteriologically pure vegetative bacteria or spores are those which are not contaminated with other bacteria or spores.
  • Microbiological techniques for obtaining such pure cultures are will known in the art. Typically single colonies are picked and spread upon an agar nutrient medium, separating colonies so that new colonies arise that are the progeny of single cells. This process is typically repeated to ensure pure cultures. Alternatively, liquid cultures can be serially diluted and plated for single colony formation. Serial repetition is desirable to ensure colony formation from single cells. See, e.g., J. H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, NY, 1972.
  • Spores, liposomase, liposomase conjugates, and nucleic acids encoding liposomase or fusion proteins of it can be administered to a tumor-bearing mammal by any means which will afford access to the tumor.
  • Spores can be injected intravenously, intradermally, subcutaneously, intramuscularly, intraperitoneally, intratrumorally, intrathecally, surgically, etc.
  • Preferred techniques are intravenous and intratumoral injections.
  • Tumor bearing mammals can be, for example, humans, pets, such as dogs and cats, agricultural animals such as cows, sheep, goats and pigs, and laboratory animals, such as rats, hamsters, monkeys, mice, and rabbits.
  • the present delivery system is useful for protein therapy or gene therapy or a combination thereof.
  • the liposomase can be provided as a protein or as a polynucleotide encoding lipsomase.
  • the anti-tumor drug or biological agent may be a protein or a polynucleotide, as well as a small chemical entity, for example, not a biological polymer.
  • Polynucleotides can be provided in a viral or non-viral vector. Any vectors which are known in the art can be used, without limitation.
  • the vector constructs may contain a tumor-specific promoter to enhance the specificity of the treatment. Examples of such promoters are known in the art.
  • CXCR4 promoter is tumor-specific in melanomas; Hexokinase type II promoter is tumor-specific in lung ' cancer; TRPM4 (Transient Receptor Potential-Melastatin 4) promoter is preferentially active in prostate cancer.
  • the anti-tumor drug may be one that directly or indirectly affects the tumor. Thus, for example, it can be a protein which stimulates the native immune response to the tumor. Alternatively, it can be an antibody which mobilizes other immune system components to destroy tumor cells. Still other agents may be toxic to tumor cells. The choice of agent is well within the skill of the artisan.
  • Conjugate proteins are post-translationally linked proteins; they may be linked by chemical linking of two polypeptides, directly or through an intermediate. Alternatively, the proteins may be linked via co-translation of two coding regions within a single open reading frame. Any means for joining two polypeptides that are not linked in nature may be used.
  • One type of conjugate which can be used utilizes an antibody molecule or portion thereof as a second protein.
  • liposomase can be linked to a variable region of an antibody thus providing the liposomase with a means of targeting to a particular site in the body which expresses the antigen to which the antibody binds.
  • Linkers may be used between portions of the conjugate proteins as desired.
  • Isolated and purified liposomase protein according to the invention is a preparation in which the liposomase activity comprises at least 10 %, 25 %, 40 %, 55 %, 70 %, 85 %, or 95 % of the protein in a composition.
  • Isolated and purified liposomase- encoding polynucleotide is one which is separated from other genes of the C. novyi genome. Thus it is separated from the genome-adjacent sequences encoding hypothetical proteins NTOl CX_ 2046 and NTOl CX_ 2048. The complete genome of C. novyi-NT has been determined and is available at NCBI as NC_008593.
  • the methods and compositions of the present invention can be applied to any tumor type.
  • the principles upon which the invention relies (such as the selective ability of liposomes to penetrate through the fenestrated endothelium present in tumors and a few other organs (8, 9), and the enhanced release of contents from a liposome in the presence of liposomase,) apply to any tumor.
  • the present invention can be used to treat tumors of the gastrointestinal tract, such as stomach, intestine, colon, rectum, esophagus, tumors of the kidney, breast, lung, liver, head and neck, and brain.
  • HCTl 16 CCL-247, human colorectal carcinoma
  • CT26 CRL-2638, murine colorectal adenocarcinoma
  • Doxorubicin was purchased from Bedford Laboratories, Bedford, OH.
  • PEGylated liposomal doxorubicin (DOXIL ® ) was purchased from Tibotec Therapeutics, Raritan, NJ.
  • EPC Chicken Egg L-cc-Phosphatidylcholine
  • HEPC Hydrogenated Chicken Egg L- ⁇ -Phosphatidylcholine
  • DSPE- PEG2000 1 ,2-Distearoyl-sn- Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000]
  • Cholesterol (Choi) were purchased from Avanti Polar Lipids, Alabaster, AL.
  • Irinotecan HCl (Camptosar) was purchased from Pharmacia & Upjohn Co., Kalamazoo, MI. Calcimycin A23187 and Triolein were obtained from Sigma (St. Louis, MO). l ⁇ -dioleoyl- ⁇ -pyrenedecanoyl-rac-glycerol (DPG) was obtained from MarkerGene Technologies (Eugene, OR). A set of nine purified lipases were purchased from Fluka, Switzerland. C. novyi-NT spores were prepared as previously described (J).
  • This lipid suspension was extruded 10 times through a double stack of 0.1 um Nuclepore filters (Whatman, Florham Park, NJ) using a Lipex Thermobarrel Extruder (Northern Lipids, Vancouver, BC, Canada).
  • the resulting colloidal suspension of Single Unilamellar Vesicles (SUV) was filter- sterilized then dialysed against 300 mM sucrose at 4°C to exchange the external milieu of the liposomes.
  • Liposomal CPT-Il Liposomal CPT-Il.
  • CPT-11 was actively loaded into liposomes using a MnSO 4 pH gradient loading method (2-4).
  • Irinotecan was mixed with liposomes at a drug:lipid molar ratio of 1 :3 and incubated at 65°C for 10 min.
  • Calcimycin was then added at a ratio of 1 ⁇ g Calcimycin: 10 ⁇ mol lipids and the suspension incubated at 65°C for 45 min.
  • the drug-loaded liposomes were then filter-sterilized and dialyzed against 300 mM sucrose at 4°C to remove unencapsulated CPT-11. Dialysis was performed in the dark to minimize photo-degradation of the drug.
  • Encapsulation efficiency was typically >99% as determined by disruption of liposomes with 1-butanol and fiuorometric measurement (excitation at 390 nm, emission at 460 nm) using a fluorescence plate reader (Fluostar Galaxy, BMG LabTech, GmbH). Concentrations were derived by reference to a CPT-1 1 standard curve.
  • mice Animal models. All animal experiments were overseen and approved by the Animal Welfare Committee of Johns Hopkins University and were in compliance with University standards. Six- to 8-week old mice purchased from Harlan Breeders, IN, were used for tumor implantation studies. Balb/c mice were used to establish CT26 tumors and athymic nu/nu mice were used to establish HCTl 16 xenografts. A minimum of five animals were used for each experimental arm. Five million tumor cells were injected subcutaneously into the right flank of each mouse and allowed to grow for ⁇ 10 days before randomization and treatment. C. novyi-NT spores were administered as a bolus tail vein injection of 300 million spores suspended in 0.2 ml phosphate buffered saline.
  • Free and liposomal drugs in the relevant arms were administered 16 hours later via the same route.
  • the doses of Doxil, doxorubicin, liposomal irinotecan and irinotecan were 10 mg/kg and 25 mg/kg, respectively.
  • Tumor volume was calculated as length x width 2 x 0.5.
  • HCTl 16 xenografts were established in athymic nu/nu mice and treated as described above. Tissue samples were harvested at various times after treatment, suspended in 70% ethanol, 0.3 N HCl and homogenized (Ultra- Turrax® T25 Basic, IKA, NC) to extract doxorubicin. Following centrifugation, doxorubicin fluorescence (excitation at 470 nm, emission at 590 nm) in the supernatant was measured with a fluorescence plate reader (FluoStar Galaxy, BMG LabTech, GmbH). Concentrations were derived by reference to a doxorubicin standard curve.
  • the pellet was solubilized in TN buffer (100 mM Tris-HCl, pH 7.5, 0.1 M NaCl) and filter-sterilized. All subsequent chromatography was performed on an AKTA Purifier FPLC system (Amersham Biosciences, Piscataway, NJ). The filtered sample was loaded onto a mono Q 5/50 GL (Amersham) column equilibrated in TN buffer. The proteins were eluted with a linear gradient of ten column volumes formed from TN buffer and 100 mM Tris-HCl 7.5, 1 M NaCl. The fractions collected were assayed for doxorubicin releasing activity as described above.
  • TN buffer 100 mM Tris-HCl, pH 7.5, 0.1 M NaCl
  • the two most active fractions from the mono Q column were pooled and loaded onto a HiLoad 16/60 Superdex 200 (Amersham) column equilibrated in 100 mM Tris-HCl pH 7.5, 0.5 M NaCl.
  • the column was isocratically eluted with the same buffer over 1.5 column volumes.
  • the fractions were assayed for doxorubicin disrupting activity as described above.
  • Proteins in the two most active fractions were separated by electrophoresis through an SDS- polyacrylamide gel and silver-stained using the SilverSNAP Stain Kit II (Pierce, Rockford, IL).
  • the single dominant band ( ⁇ 45 kD) was excised for analysis by LC/MS/MS.
  • LC/MS/MS Peptide Analysis The excised protein band was digested in-gel with trypsin, and analyzed as previously described (7). In brief, purified tryptic fragments were injected onto a 150 x 0.3 mm Vydac reverse phase column at 10 ⁇ L/min for 5 min in 5% buffer B using an Agilent 1100 Series Capillary-LC system. Buffers A (0.1% acetic acid) and B (99.9% acetonitrile and 0.1% acetic acid) were employed in the liquid chromatography (LC) step of the LC/MS/MS analysis. Peptides were eluted from the column with a 10-65% buffer B gradient over 90 minutes at a rate of 2 ⁇ L/min.
  • LC liquid chromatography
  • Eluted peptides were detected by a LCQ DECA XP mass spectrometer (Thermo, MA), equipped with an electrospray ionization source, a low flow metal needle assembly operating in data dependent mode.
  • the method consisted of two scan events, a full scan and a second data dependent MS/MS scan.
  • the dynamic mass range of the full scan was set at 300 to 3000 ifa/z.
  • the resulting MS/MS data dependent scan rejected known 'contaminant' masses of 371.0, 391.0, 445.0, 462.0, 1221.89, 1321.9, 1421.9, 1521.8, 1521.9, 1621.9, 1721.9 and 1821.9 m/z.
  • the pLip plasmid was digested with SnaB I to excise a 45 bp fragment containing the serine residue to be mutated.
  • the pLip(S127G) plasmid was generated by blunt-end ligating the digested plasmid with a replacement 45 bp double-stranded oligonucleotide of sequence 5'- GTAAAGTTCATTTAATAGGACACGGTCAA GGTGGACAAACTATAC (SEQ ID NO: 5).
  • the plasmid pLip(S127X) plasmid was generated in the same fashion using the double-stranded oligonucleotide 5'-GTAAAGTTCATTTAATAGGACA CTAATAAGGTGGACAAACTATAC (SEQ ID NO: 6). The targeted alterations (underlined above) and orientation of the inserts were verified by DNA sequencing. [49] Protein Expression. The expression vectors described above were transformed by heat shock into the Rosetta-gami(DE3)pLysS strain of E. coli (Novagen, Madison, WI).
  • Lysates were prepared by sonication in a Bioruptor sonicating bath (Diagenode, Belgium) and centrifugation at ⁇ 14,000 g was performed to remove insoluble matter.
  • Lipase Activity units were derived by reference to a Pseudomonas cepacia lipase standard curve. 1 unit (U) corresponds to the equivalent amount of P. cepacia lipase activity which liberates 1 ⁇ mol per minute of oleic acid from triolein.
  • Doxil is a liposomal formulation which encapsulates doxorubicin, a DNA- damaging agent and widely used chemotherapeutic agent. Liposome-encapsulated doxorubicin has been shown to result in improved outcomes compared to unencapsulated doxorubicin in a variety of experimental and clinical studies (18-21).
  • the liposomes in Doxil are surface modified by PEGylation to increase their circulation time (18).
  • treatment with C. novyi-NT spores alone resulted in germination and necrosis within the centrally hypoxic region of the tumor, but left a well-oxygenated viable rim that eventually regrew (Fig. IA).
  • doxorubicin nor Doxil alone resulted in prolonged therapeutic effects in these mice.
  • the effects were remarkable, resulting in complete regression of tumors in all mice (Fig. IA) and cures in more than half of them (Fig. IB).
  • mice treated with C. novyi-NT and free doxorubicin at the same dose exhibited dramatic morbidity, with 100% of mice dying within 2 weeks, emphasizing the crucial role of liposomal encapsulation in reducing systemic toxicity (18).
  • doxorubicin found in infected tumors had been released from liposomes and was bound to tumor cell nuclei, as revealed by immunofluorescence (Fig. 2B).
  • concentration of doxorubicin in conventionally-treated tumors was previously shown to be higher and more stable after administration of Doxil than after administration of doxorubicin (22).
  • the administration of C. novyi-NT spores plus Doxil resulted in a greater than 100-fold increase in tumor drug exposure compared to that achieved with an equivalent dose of free doxorubicin, without increasing drug concentrations in normal tissues.
  • novyi-NT genome contains four genes predicted to encode phospholipases (26).
  • One of these four genes (NT01CX0979) encoded an extracellular phospholipase C protein (23, 25) which was expressed at high levels in growing bacteria (26).
  • novyi-NT genome as the reference, the polypeptide was found to be encoded by NTOl CX2047, a putative lipase.
  • the two extracellular lipases identified in the C. novyi-NT genome (NT01CX0630 and NT01CX2047) were not highly homologous to each other (47% . aa identity) or to their closest counterparts in other bacteria (50 ⁇ 55% aa identity to a C. tetani lipase).
  • the identification of the liposome-disrupting factor as the product of NT01CX2047 was consistent with information from the genomic analysis of C. novyi- NT, which revealed that NT01CX2047 was preferentially expressed in late log phase, was predicted to be extracellular, and was highly expressed in tumors after infection with C. novyi-NT (26).
  • NT01CX2047-expressing clones exhibited lipase activity, whereas clones cured of the vector (described below) did not (Fig. 4B).
  • a derivative was generated in which a stop codon was substituted for the serine residue at amino acid 127 (S 127X).
  • S 127G was replaced by glycine.
  • S 127 is found within the highly-conserved GXSXG lipase motif and was predicted to be the essential catalytic serine responsible for lipase activity (27).
  • both the S127G missense mutant and the S127X truncation mutant were devoid of lipase activity though each produced similar amounts of NT01CX2047 polypeptide (Fig. 4A, B).
  • the non-catalytic S127G mutant exhibited the same poor growth as wild-type NT01CX2047, whereas the S127X mutant grew much more robustly.
  • EXAMPLE 8 genetic separation of lipase activity and liposome-disrupting activity
  • liposomes containing CPT-Il irinotecan
  • CPT-Il a topoisomerase inhibitor that, like doxorubicin
  • Single doses of liposomal CPT-I l were injected alone or 16 hours after C. novyi-NT administration, just as in the Doxil experiments.
  • Both tumor types CT26 in syngeneic mice and human colorectal cancer cell xenografts in nude mice
  • all tumors regressed and long-term cures were achieved in >60% of the mice (Fig. 5).
  • doxorubicin from Doxil in tumors could theoretically be due to other enzymes released from C. novyi-NT or infected tumor cells.
  • the major Doxil-disrupting factor secreted from C. novyi-NT is liposomase (Fig. 3) and its gene is expressed at high levels in infected tumors (26), suggesting that liposomase significantly contributes to the in vivo effects.
  • liposomase has been identified and cloned, it can be incorporated into other anticancer strategies. These strategies include cancer gene therapy employing viral or non-viral delivery systems and antibody-directed enzyme prodrug therapy (3, 4, 31, 32).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Mycology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)
PCT/US2007/014274 2006-06-19 2007-06-19 Tumor-specific delivery of therapeutic agents via liposomase WO2007149433A2 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
KR1020087031596A KR101376634B1 (ko) 2006-06-19 2007-06-19 리포좀아제에 의한 치료제의 종양­특이적 전달
CN200780027001.3A CN101489593B (zh) 2006-06-19 2007-06-19 通过脂质体酶的治疗剂肿瘤特异性递送
JP2009516542A JP5319523B2 (ja) 2006-06-19 2007-06-19 リポソマーゼによる治療物質の腫瘍特異的送達
US12/304,178 US8444963B2 (en) 2006-06-19 2007-06-19 Tumor specific delivery of therapeutic agents via liposomase
US13/692,064 US8568708B2 (en) 2006-06-19 2012-12-03 Tumor-specific delivery of therapeutic agents via liposomase
US13/963,387 US8901100B2 (en) 2006-06-19 2013-08-09 Tumor-specific delivery of therapeutic agents via liposomase
US14/556,890 US20150306184A1 (en) 2006-06-19 2014-12-01 Tumor-Specific Delivery of Therapeutic Agents via Liposomase/US

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US81454606P 2006-06-19 2006-06-19
US60/814,546 2006-06-19

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US12/304,178 A-371-Of-International US8444963B2 (en) 2006-06-19 2007-06-19 Tumor specific delivery of therapeutic agents via liposomase
US13/692,064 Division US8568708B2 (en) 2006-06-19 2012-12-03 Tumor-specific delivery of therapeutic agents via liposomase

Publications (2)

Publication Number Publication Date
WO2007149433A2 true WO2007149433A2 (en) 2007-12-27
WO2007149433A3 WO2007149433A3 (en) 2008-11-06

Family

ID=38834068

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/014274 WO2007149433A2 (en) 2006-06-19 2007-06-19 Tumor-specific delivery of therapeutic agents via liposomase

Country Status (5)

Country Link
US (4) US8444963B2 (enrdf_load_stackoverflow)
JP (1) JP5319523B2 (enrdf_load_stackoverflow)
KR (1) KR101376634B1 (enrdf_load_stackoverflow)
CN (1) CN101489593B (enrdf_load_stackoverflow)
WO (1) WO2007149433A2 (enrdf_load_stackoverflow)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012054717A3 (en) * 2010-10-21 2012-06-28 The Johns Hopkins University Detecting and treating solid tumors through selective disruption of tumor vasculature
WO2021123391A1 (en) 2019-12-18 2021-06-24 Exomnis Biotech B.V. Genetically modified clostridium strains and uses thereof

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2601934A1 (en) * 2009-01-22 2013-06-12 Ludwig-Maximilians-Universität München Vesicular phospholipid gels comprising proteinaceous substances
WO2014010758A1 (ja) 2012-07-13 2014-01-16 学校法人帝京平成大学 抗腫瘍剤、腫瘍検出用マーカー及び経口ワクチン剤
WO2016141167A1 (en) 2015-03-03 2016-09-09 Cureport, Inc. Combination liposomal pharmaceutical formulations
WO2016141161A1 (en) 2015-03-03 2016-09-09 Cureport, Inc. Dual loaded liposomal pharmaceutical formulations
WO2017061562A1 (ja) * 2015-10-07 2017-04-13 塩水港精糖株式会社 タキサン化合物を内包するリポソーム
TW202345881A (zh) * 2022-03-23 2023-12-01 國立研究開發法人理化學研究所 用以治療惡性腫瘤的組合醫藥

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
FI20031528A0 (fi) * 2003-10-17 2003-10-17 Ctt Cancer Targeting Tech Oy Terapeuttinen liposomikoostumus ja menetelmä sen valmistamiseksi
JP2005298486A (ja) 2004-03-15 2005-10-27 Nipro Corp リポソームを含む癌治療用医薬組成物
US7740861B2 (en) * 2004-06-16 2010-06-22 University Of Massachusetts Drug delivery product and methods
AU2005298364B2 (en) 2004-10-26 2009-03-26 Ortho Biotech Products L.P. Pegylated Liposomal Doxorubicin in combination with Ecteinescidin 743

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DIAZ ET AL.: 'Pharmacologic and Toxicologic Evaluation of C. noyi-NT Spores' TOXICOLOGICAL SCIENCES vol. 88, no. 2, 2005, pages 562 - 575 *
MELLAERT ET AL.: 'Clostridium spores as anti-tumour' TRENDS IN MICROBIOLOGY vol. 14, no. 4, April 2006, pages 190 - 196, XP005387792 *
THURSTON ET AL.: 'Cationic Liposomes Target Angiogenic Endothelial Cells in Tumors and Chronic Inflammation in Mice' J. CLIN. INVEST. vol. 101, no. 7, April 1998, pages 1401 - 1413, XP000863045 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012054717A3 (en) * 2010-10-21 2012-06-28 The Johns Hopkins University Detecting and treating solid tumors through selective disruption of tumor vasculature
WO2021123391A1 (en) 2019-12-18 2021-06-24 Exomnis Biotech B.V. Genetically modified clostridium strains and uses thereof

Also Published As

Publication number Publication date
US8568708B2 (en) 2013-10-29
CN101489593B (zh) 2015-11-25
JP5319523B2 (ja) 2013-10-16
CN101489593A (zh) 2009-07-22
US20130142862A1 (en) 2013-06-06
US8901100B2 (en) 2014-12-02
US20130315986A1 (en) 2013-11-28
KR101376634B1 (ko) 2014-03-27
US20110250258A1 (en) 2011-10-13
US20150306184A1 (en) 2015-10-29
JP2009541319A (ja) 2009-11-26
US8444963B2 (en) 2013-05-21
WO2007149433A3 (en) 2008-11-06
KR20090027674A (ko) 2009-03-17

Similar Documents

Publication Publication Date Title
US8901100B2 (en) Tumor-specific delivery of therapeutic agents via liposomase
JP5711099B2 (ja) シスプラチン及び他の薬剤又はリポソーム中に封入された遺伝子を用いてのヒト癌のための療法
JP5722782B2 (ja) ナノエマルジョン治療用組成物及びその使用方法
CN113164589A (zh) 用于调节单核细胞和巨噬细胞发炎表型的组合物和方法以及其免疫疗法用途
HU229805B1 (en) Photochemical internalization for delivery of molecules into the cytosol
EA024826B1 (ru) Способ лечения латентного туберкулеза
AU2021231914A1 (en) Compositions and methods for the treatment of intracellular bacterial infections
US10668172B2 (en) Treatment for exposure to nerve agent
CA2802351A1 (en) Clostridium taeniosporum spores and spore appendages as surface display hosts, drug delivery devices, and nanobiotechnological structures
CN120500329A (zh) 用于治疗剂的靶向递送的自组装脂质纳米颗粒
JP2020536968A (ja) 共生疾患進行に対する包括的ワクチン設計
AU2021231086A1 (en) Compositions and methods for the treatment of intracellular bacterial infections
US20250249079A1 (en) Compositions and methods for the treatment of actinomycetia infections
US11851464B2 (en) Methods and compositions related to recombinant NEIL2
WO2023200865A2 (en) Cancer therapy
WO2024233406A2 (en) Cpmv-rna immunotherapy
JP2023542341A (ja) 標的化された抗原送達システム及びその使用

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200780027001.3

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07809666

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 2009516542

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 1020087031596

Country of ref document: KR

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 07809666

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 12304178

Country of ref document: US